Die Rolle von DNMT1 bei der postmitotischen Entwicklung von inhibitorischen kortikalen Interneuronen

GABAerge Interneurone modulieren die Signalweiterleitung im Neokortex und Defekte in ihrer Entwicklung zum Beispiel durch Veränderung in der Genexpression sind mit diversen Erkrankungen assoziiert. Epigenetische Modifikationen, wie DNA-Methylierungen und Histonmodifikationen ermöglichen die Regulation einer spezifischen Genaktivität. Welche Rolle die DNA-Methyltransferase 1 (DNMT1) bei der postmitotischen Entwicklung von kortikalen Interneuronen spielt, ist eine der zentralen Fragen dieser Arbeit. Hierfür wurden Untersuchungen am konditionalen Knockout-Mausmodel durchgeführt, in dem Dnmt1 in postmitotischen, Hmx3-abstammenden Interneuronen deletiert wurde. Die Wanderung unreifer Interneurone von ihren subpallialen Ursprungsorten in kortikale Bereiche erfordert die Ausbildung und den Erhalt einer typischen migratorischen Morphologie, welche sich nach Deletion von Dnmt1 gestört zeigte. Vergleichende Transkriptomanalysen stellten eine erhöhte Expression von Pak6 in Dnmt1-defizienten Zellen heraus, welche zu den morphologischen Veränderungen beitrug. Weitere Analysen zu potentiell durch DNMT1 regulierten Genen rückten den Transkriptionsfaktor LHX1 in den Fokus dieser Arbeit. Zur Untersuchung seiner Funktion wurde ein Knockout-Mausmodel generiert, in dem Lhx1 in Hmx3-abstammenden Interneuronen deletiert wurde. Phänotypische Analysen von Mausstadien, sowie in vitro Studien an kultivierten Primärzellen und N2a-Zellen zeigten, dass LHX1 das zelluläre Überleben begrenzt. Jedoch konnte weder für Pak6 noch für Lhx1 eine direkte Transkriptionskontrolle durch DNMT1-vermittelte DNA-Methylierung herausgestellt werden. Es zeigte sich hingegen eine DNMT1-abhängende Assoziation der repressiven Histonmarkierung H3K27me3 an Pak6 und Lhx1 Genregionen, welche die Repression beider Gene vermitteln könnte. Insgesamt zeigen die dargestellten Ergebnisse, dass eine durch DNMT1 vermittelte komplexe Genregulation essentiell für die Migration und Vitalität von POA-abstammenden Interneuronen ist

Palmitate-Triggered COX2/PGE2-Related Hyperinflammation in Dual-Stressed PdL Fibroblasts Is Mediated by Repressive H3K27 Trimethylation

The interrelationships between periodontal disease, obesity-related hyperlipidemia and mechanical forces and their modulating effects on the epigenetic profile of periodontal ligament (PdL) cells are assumed to be remarkably complex. The PdL serves as a connective tissue between teeth and alveolar bone and is involved in pathogen defense and the inflammatory responses to mechanical stimuli occurring during tooth movement. Altered inflammatory signaling could promote root resorption and tooth loss. Hyperinflammatory COX2/PGE2 signaling was reported for human PdL fibroblasts (HPdLFs) concomitantly stressed with Porphyromonas gingivalis lipopolysaccharides and compressive force after exposure to palmitic acid (PA). The aim of this study was to investigate the extent to which this was modulated by global and gene-specific changes in histone modifications. The expression of key epigenetic players and global H3Kac and H3K27me3 levels were quantitatively evaluated in dual-stressed HPdLFs exposed to PA, revealing a minor force-related reduction in repressive H3K27me3. UNC1999-induced H3K27me3 inhibition reversed the hyperinflammatory responses of dual-stressed PA cultures characterized by increased COX2 expression, PGE2 secretion and THP1 adhesion. The reduced expression of the gene encoding the anti-inflammatory cytokine IL-10 and the increased presence of H3K27me3 at its promoter-associated sites were reversed by inhibitor treatment. Thus, the data highlight an important epigenetic interplay between the different stimuli to which the PdL is exposed

Hyperlipidemic Conditions Impact Force-Induced Inflammatory Response of Human Periodontal Ligament Fibroblasts Concomitantly Challenged with P. gingivalis -LPS

In obese patients, enhanced serum levels of free fatty acids (FFA), such as palmitate (PA) or oleate (OA), are associated with an increase in systemic inflammatory markers. Bacterial infection during periodontal disease also promotes local and systemic low-grade inflammation. How both conditions concomitantly impact tooth movement is largely unknown. Thus, the aim of this study was to address the changes in cytokine expression and the secretion of human periodontal ligament fibroblasts (HPdLF) due to hyperlipidemic conditions, when additionally stressed by bacterial and mechanical stimuli. To investigate the impact of obesity-related hyperlipidemic FFA levels on HPdLF, cells were treated with 200 µM PA or OA prior to the application of 2 g/cm(2) compressive force. To further determine the additive impact of bacterial infection, HPdLF were stimulated with lipopolysaccharides (LPS) obtained from Porphyromonas gingivalis. In mechanically compressed HPdLF, PA enhanced COX2 expression and PGE2 secretion. When mechanically stressed HPdLF were additionally stimulated with LPS, the PGE2 and IL6 secretion, as well as monocyte adhesion, were further increased in PA-treated cultures. Our data emphasize that a hyperlipidemic condition enhances the susceptibility of HPdLF to an excessive inflammatory response to compressive forces, when cells are concomitantly exposed to bacterial components

Immunometabolic capacities of nutritional fatty acids in regulation of inflammatory bone cell interaction and systemic impact of periodontal infection

IntroductionNovel preventive strategies in periodontal disease target the bacterial-induced inflammatory host response to reduce associated tissue destruction. Strategies focus on the modulation of tissue-destroying inflammatory host response, particularly the reduction of inflammation and promotion of resolution. Thereby, nutrition is a potent immunometabolic non-pharmacological intervention. Human studies have demonstrated the benefit of olive oil-containing Mediterranean-style diets (MDs), the main component of which being mono-unsaturated fatty acid (FA) oleic acid (OA (C18:1)). Hence, nutritional OA strengthened the microarchitecture of alveolar trabecular bone and increased circulating pro-resolving lipid mediators following bacterial inoculation with periodontal pathogen Porphyromonas gingivalis, contrary to saturated FA palmitic acid (PA (C16:0)), which is abundant in Western-style diets. Additionally, the generalized distribution of inflammatory pathway mediators can occur in response to bacterial infection and compromise systemic tissue metabolism and bone homeostasis distant from the side of infection. Whether specific FA-enriched nutrition and periodontal inoculation are factors in systemic pathology that can be immune-modulatory targeted through dietary substitution is unknown and of clinical relevance.MethodsNormal-weight C57BL/6-mice received OA-or PA-enriched diets (PA-ED, OA-ED, PA/OA-ED) or a normal-standard diet (n=12/group) for 16 weeks and were orally infected with P. gingivalis/placebo to induce periodontal disease. Using histomorphometry and LC-MS/MS, systemic bone morphology, incorporated immunometabolic FA-species, serological markers of bone metabolism, and stress response were determined in addition to bone cell inflammation and interaction in vitro.ResultsIn contrast to OA-ED, PA-ED reduced systemic bone microarchitecture paralleled by increased lipotoxic PA-containing metabolite accumulation in bone. Substitution with OA reversed the bone-destructive impact of PA, which was accompanied by reduced diacylglycerols (DAG) and saturated ceramide levels. Further, PA-associated reduction in mineralization activity and concomitant pro-inflammatory activation of primary osteoblasts were diminished in cultures where PA was replaced with OA, which impacted cellular interaction with osteoclasts. Additionally, PA-ED increased osteoclast numbers in femurs in response to oral P. gingivalis infection, whereas OA-ED reduced osteoclast occurrence, which was paralleled by serologically increased levels of the stress-reducing lipokine PI(18:1/18:1).ConclusionOA substitution reverses the bone-destructive and pro-inflammatory effects of PA and eliminates incorporated lipotoxic PA metabolites. This supports Mediterranean-style OA-based diets as a preventive intervention to target the accumulation of PA-associated lipotoxic metabolites and thereby supports systemic bone tissue resilience after oral bacterial P. gingivalis infection

Hyperlipidemic Conditions Impact Force-Induced Inflammatory Response of Human Periodontal Ligament Fibroblasts Concomitantly Challenged with P. gingivalis-LPS

In obese patients, enhanced serum levels of free fatty acids (FFA), such as palmitate (PA) or oleate (OA), are associated with an increase in systemic inflammatory markers. Bacterial infection during periodontal disease also promotes local and systemic low-grade inflammation. How both conditions concomitantly impact tooth movement is largely unknown. Thus, the aim of this study was to address the changes in cytokine expression and the secretion of human periodontal ligament fibroblasts (HPdLF) due to hyperlipidemic conditions, when additionally stressed by bacterial and mechanical stimuli. To investigate the impact of obesity-related hyperlipidemic FFA levels on HPdLF, cells were treated with 200 µM PA or OA prior to the application of 2 g/cm2 compressive force. To further determine the additive impact of bacterial infection, HPdLF were stimulated with lipopolysaccharides (LPS) obtained from Porphyromonas gingivalis. In mechanically compressed HPdLF, PA enhanced COX2 expression and PGE2 secretion. When mechanically stressed HPdLF were additionally stimulated with LPS, the PGE2 and IL6 secretion, as well as monocyte adhesion, were further increased in PA-treated cultures. Our data emphasize that a hyperlipidemic condition enhances the susceptibility of HPdLF to an excessive inflammatory response to compressive forces, when cells are concomitantly exposed to bacterial components

Regulation of neuronal survival by DNA methyltransferases

The limited regenerative capacity of neuronal cells requires tight orchestration of cell death and survival regulation in the context of longevity, age-associated diseases as well as during the development of the nervous system. Subordinate to genetic networks epigenetic mechanisms like DNA methylation and histone modifications are involved in the regulation of neuronal development, function and aging. DNA methylation by DNA methyltransferases (DNMTs), mostly correlated with gene silencing, is a dynamic and reversible process. In addition to their canonical actions performing cytosine methylation, DNMTs influence gene expression by interactions with histone modifying enzymes or complexes increasing the complexity of epigenetic transcriptional networks. DNMTs are expressed in neuronal progenitors, post-mitotic as well as adult neurons. In this review, we discuss the role and mode of actions of DNMTs including downstream networks in the regulation of neuronal survival in the developing and aging nervous system and its relevance for associated disorders

Oleic acid-related anti-inflammatory effects in force-stressed PdL fibroblasts are mediated by H3 lysine acetylation associated with altered IL10 expression

The initiation of a spatially and temporally limited inflammation is essential for tissue and bone remodelling by the periodontal ligament (PdL) located between teeth and alveolar bone. Nutritional components may cause alterations in the inflammatory response of PdL fibroblasts to mechanical stress such as those occurring during orthodontic tooth movement (OTM). Recently, we reported an attenuated pro-inflammatory response of human PdL fibroblasts (HPdLFs) to compressive forces when stimulated with oleic acid (OA), a monounsaturated fatty acid particularly prominent in the Mediterranean diet. Fatty acids could serve as alternative source of acetyl-CoA, thereby affecting epigenetic histone marks, such as histone 3 lysine acetylation (H3Kac) in a lipid metabolism-dependent manner. In this study, we aimed to investigate the extent to which OA exerts its anti-inflammatory effect in compressed HPdLFs via changes in H3Kac. Six-hour compressed HPdLFs showed increased H3Kac when cultured with OA. Inhibition of histone deacetylases resulted in a comparable IL10-increase as observed in compressed OA-cultures. In contrast, inhibition of histone acetyltransferases, particularly p300/CBP, in compressed HPdLFs exposed to OA normalized the inflammatory response to control levels. OA-dependent increased association of H3Kac to IL10 promoter regions in compressed HPdLFs further strengthened the assumption that OA exhibits its anti-inflammatory properties via modulation of this epigenetic mark. In conclusion, our study strongly suggests that nutritional components can directly affect PdL cells via changes in their epigenetic code. Since epigenetic inhibitors are already widely used clinically, they may hold promise for novel approaches for personalized orthodontic treatment that incorporates nutritional and metabolism-related changes